Background: Polyethylene glycols (PEGs) are polymers of varying molecular weight (MW) used widely as excipients in drugs and other products, including the mRNA vaccines against coronavirus disease 2019. Allergy to PEGs is rare. Skin testing and graded challenge carries a high risk of inducing systemic reactions. Objective: We evaluated skin prick test (SPT) results and in vitro reactivity over time to different MW PEGs and assessed cross-sensitization patterns in PEG allergy. Methods: Ten patients with previously diagnosed PEG allergy underwent SPT twice with PEGs 26 months apart. Lower MW (PEG 300, 3000, 6000) were tested, followed by PEG 20,000, in stepwise, increasing concentrations. Cross-sensitization to polysorbate 80 and poloxamer 407 was assessed. SPT was performed in 16 healthy controls. In vitro basophil histamine release (HR) test and passive sensitization HR test were performed in patients and controls. Results: Patients previously testing positive on SPT to PEG 3000 and/or 6000 also tested positive to PEG 20,000. Patients with a longer interval since diagnosis tested negative to lower MW PEGs and positive mainly to higher concentrations of PEG 20,000. Three patients developed systemic urticaria during SPT. Eight patients showed cross-sensitization to poloxamer 407 and 3 to polysorbate 80. All controls tested negative. In vitro tests showed limited usefulness. Conclusions: Skin test reactivity to PEG can decrease over time, but titrated SPT with increasing concentrations of PEG 20,000 can be diagnostic when lower MW PEGs test negative. To avoid systemic reactions, stepwise SPT is mandatory. (J Allergy Clin Immunol 2021;nnn:nnn-nnn.)

Antigen-mediated mast cell activation is a pivotal step in the initiation of allergic disorders including anaphylaxis and atopy. To date, studies aimed at investigating the mechanisms regulating these responses, and studies designed to identifying potential ways to prevent them, have primarily been conducted in rodent mast cells. However, to understand how these responses pertain to human disease, and to investigate and develop novel therapies for the treatment of human mast cell-driven disease, human mast cell models may have greater relevance. Recently, a number of systems have been developed which allow investigators to readily obtain sufficient quantities of human mast cells to conduct these studies. These mast cells release the appropriate suite of inflammatory mediators in response to known mast cell activators including antigen. These systems have also been employed to examine the signaling events regulating these responses. Proof of principle studies have also demonstrated utility of these systems for the identification of potential inhibitors of mast cell activation and growth. In this unit, we describe techniques for the development and culture of human mast cells from their progenitors and the culture of human mast cell lines. The relative merits and drawbacks of each model are also described.

IL-33, an IL-1 family member and ligand for the IL-1 receptor-related protein ST2, has been associated with induction of Th2 cytokines such as IL-4, IL-5, and IL-13. Here, we report that IL-33 can initiate IL-9 protein secretion in vitro in human CD4+ T cells and basophils isolated from peripheral blood. TGF-β has been described as a critical factor for IL-9 induction in Th2 cells; however, we found that TGF-β also induces co-production of IL-9 in purified, naïve (>99%) CD4+CD45RA+CD45RO−CD25− T cells differentiated towards a Th1 profile. Subsequently, it was demonstrated that TGF-β is important, although not an absolute requirement, for IL-9 production in CD4+ T cells. IL-9 production by purified (>95%) human basophils, cultured for 24 h with IL-3 or IL-33, was found, with a strong synergy between the two, likely to be explained by the IL-3 upregulated ST2 expression. Collectively, these data indicate that barrier functioning cells are important for the regulation of IL-9 production by immune cells in inflamed tissue.

The transmembrane adaptor protein (TRAP), NTAL, is phosphorylated in mast cells following FcvarepsilonRI aggregation whereby it cooperates with LAT to induce degranulation. The Kit ligand, stem cell factor (SCF), enhances antigen-induced degranulation and this also appears to be NTAL-dependent. However, Kit and FcvarepsilonRI appear to utilize different mechanisms to induce NTAL phosphorylation. Thus, we examined whether the responsible kinases selectively phosphorylated distinct tyrosines in NTAL and explored the implications for downstream signaling. Whereas FcvarepsilonRI required Lyn and Syk for NTAL phosphorylation, Kit appeared to directly phosphorylate NTAL. Furthermore, co-transfection studies with NTAL constructs revealed that Lyn, Syk, and Kit phosphorylate different tyrosines in NTAL. The tyrosines principally phosphorylated by Syk were recognized as Grb2-binding sites, whereas Lyn and Kit phosphorylated other tyrosines, both inside and outside of these motifs. Pull down studies revealed that PLCgamma1 associated with the two terminal Syk-phosphorylated Grb2-binding sites, which would help to explain the observed decrease in antigen-induced calcium signal and degranulation in NTAL-knock down-human mast cells. The observations reported herein support the conclusion that NTAL may be differentially utilized by specific receptors for relaying alternative signals and this suggests a flexibility in the function of TRAPs not previously appreciated.